Spontaneous recovery occurs in a minority of patients with acute hepatitis C, but is associated with vigorous and long-lasting cellular immune responses (J. Exp. Med. 2001; 194:1395, Nat. Med. 2000; 6: 578). In contrast, treatment-induced recovery from hepatitis C can be achieved in the majority of patients who are treated during the acute phase of hepatitis C (NEJM 2001; 20: 1452), but the mechanisms of viral clearance and their effect on cellular immune responsiveness are not known. Both direct effects, such as immune modulation of Th2 to Th1 responses, and indirect effects, such as prevention of downregulation and exhaustion of cellular responses by rapid and early reduction of viral load, have been proposed. To investigate how early antiviral therapy and successful reduction of HCV load affect HCV-specific T cell responses, we performed detailed, prospective clinical, immunological and virological studies on patients with acute hepatitis C who received antiviral therapy and were followed over a period of 1-2 years. This comprehensive analysis demonstrated that none of patients suffered from a primary failure of the HCV-specific cellular immune response in the acute phase prior to treatment. Even in the absence of detectable T cell proliferation, multispecific IFNg+ T cell responses were found in all patients. In individual patients, phases of apparent immune control and decreased viral load were followed by phases of weaker immune responses and increased viral load. After initiation of peginterferon/ribavirin or interferon/ribavirin combination therapy (mean of 20 weeks after defined exposure to HCV), all sustained treatment responders demonstrated a rapid decrease of HCV RNA levels followed by a more gradual decrease of the HCV-specific T cell responses to undetectable level. In contrast, two patients who maintained HCV-specific T cell responses through the course of therapy did not clear HCV completely as shown by subsequent viral breakthrough. Collectively, these results suggest that HCV and antigens are cleared as a result of antiviral rather than immunomodulatory actions of interferon and ribavirin. HCV-specific T cell responses disappear from the blood if treatment is successful. Importantly, this pattern differs significantly from that reported to occur in spontaneously recovered patients, where we have reported HCV-specific T cell to persist long-term after recovery (Nature Medicine 6: 578-582, 2000). Because maintenance of cellular immune responses has been associated with immune protection upon rechallenge (J. Virol., 2003; 77: 4781), patients who resolve acute hepatitis C as a result of antiviral therapy may have less protective immunity than those who recover spontaneously. In a second project, using in vitro studies and animal studies, we investigated a viral escape mechanism that contributes to the weakness of HCV-specific T cell responses in persistent HCV infection. We hypothesized that changes in the proteasomal cleavage pattern influence the quality and quantity of epitopes presented on MHC class I molecules, thereby impair the induction of T cell responses and attenuate the cellular immune response in persistent infection. Specifically, we asked whether a frequent HCV mutation affected proteasomal epitope generation. We had observed this mutation in patients with persistent hepatitis C but not in the infectious source, and it was located adjacent to the carboxyterminus of an immunodominant HLA-A2-restricted NS3 T cell epitope. To answer this question, we performed in vitro studies using isolated proteasomes and in vivo studies using HLA-A2 transgenic mice. For in vitro analysis, constitutive proteasomes were isolated from HepG2 cells and immunoproteasomes from IFN-g-stimulated HepG2 and from MEC-217 cells transfected with the immunoproteasome subunits LMP-2, LMP-7 and MECL-1. Wildtype and mutant NS3 polypeptides were digesttted with the respective proteasomes and the digestion products were separated and analyzed by RP-HPLC and mass spectrometry. Analysis of digestion products demonstrated impaired carboxyterminal cleavage of the epitope from the mutant but not from the wildtype polypeptide. For in vivo analysis, HLA-A2-transgenic mice were infected with a recombinant vaccinia virus (VV) encoding either wildtype or mutant full-length NS3. Two weeks later, the frequency of NS3 epitope-specific CD8+ T cells in the spleen was determined by (a) ex vivo tetramer assays, (b) intracellular IFN-g staining and (c) IFN-g ELISpot assays. In each assay, the frequency of NS3-epitope-specific cells was higher in mice immunized with wildtype than in those immunized with the mutant NS3. In contrast, this difference was not observed for VV-specific T cells. Moreover, even after in vitro expansion of NS3 epitope-specific T cells, cytotoxic responses of 6 mice immunized with VV encoding wildtype NS3 remained significantly stronger than responses of 6 mice immunized with VV encoding mutant NS3 (p=0.036). In summary, this study demonstrates that a conservative HCV mutation in an epitope flanking region leads to altered antigen processing in vitro and in vivo. Interference with the induction of CD8+ T cells by alteration of proteasomal antigen processing explains the characteristic weakness of the cellular immune response in HCV infection and represents an immune escape mechanism for HCV. Finally, in a third project we studied the nature of CD4/CD8 double positive cells that we had observed in the blood of several patients with acute and chronic hepatitis C. Extending these studies to HCV recovered persons and healthy control persons we now demonstrate that the circulating CD4/CD8 double-positive cell population contains mature effector memory lymphocytes specific for antigens of multiple past, latent and high-level persistent viral infections. Upon in vitro antigenic challenge, a higher frequency of CD4/CD8 double-positive than single-positive cells displayed a Th1/Tc1 cytokine profile and proliferated. Ex vivo, more double-positive than single-positive cells exhibited a differentiated phenotype. Accordingly, their lower content of T cell receptor excision circles (TRECs) and shorter telomeres proved that they had divided more frequently than single-positive cells. Consistent with expression of the tissue-homing marker CXCR3, CD4/CD8 double-positive cells were demonstrated in situ at the site of persistent viral infection, i.e. in liver tissue of patients with chronic hepatitis C. Finally, a prospective analysis of HCV infection in a chimpanzee, the only animal model for HCV infection, showed a close correlation between the frequency of activated CD4/CD8 double-positive cells and HCV kinetics. Collectively, these findings demonstrate that peripheral CD4/CD8 double-positive cells take part in the adaptive immune response against infectious pathogens and broaden the perception of the T cell populations involved in antiviral immune responses.